Oncolytic virus

Abstract

This invention relates to gene constructs, as well as expression cassettes and oncolytic viruses containing gene constructs, and their use in methods for treating, preventing, or improving cancer. Gene constructs, expression cassettes, and oncolytic viruses are particularly useful, though not exclusively, for treating bladder cancers, such as nonmuscle-invasive bladder cancer.

Description

[Technical Field]

[0001] The present invention relates to gene constructs, expression cassettes and oncolytic viruses comprising the gene constructs, and uses thereof in methods for treating, preventing or ameliorating cancer. The gene constructs, expression cassettes and oncolytic viruses are useful, particularly but not exclusively, for treating bladder cancer such as non-muscle invasive bladder cancer. [Background Art]

[0002] Non-muscle invasive bladder cancer (NMIBC) is a highly prevalent and major health problem with a lifelong risk of recurrence. In the United Kingdom, there are approximately 10,000 new cases of NMIBC per year. Surgery is the standard treatment, but it is associated with high recurrence rates and tumor progression.

[0003] Immunotherapy with Bacillus Calmette-Guérin (BCG), a live tuberculosis vaccine, administered intravesicularly, has been the standard of care for 40 years, and is infused post-surgery in high-risk disease (intermediate and high-grade cancers). BCG acts partly as an immunotherapy through activation of receptors so-called Toll-like receptor ligands (TLRs), which are important for initiating the host's immune defense mechanisms. While this reduces the risk of recurrence, it comes at the high cost of local and systemic toxicity, which greatly limits its use. As a result, approximately one-third of patients do not respond to BCG treatment, while another third experience severe adverse effects and have to discontinue treatment.

[0004] Accordingly, there is an urgent unmet need for alternative treatments for a substantial proportion (up to 60%) of bladder cancer patients who do not respond to or cannot tolerate BCG. In particular, there is a need for new local treatments that can exploit the immunomodulatory effects of BCG, improve efficacy, but are free of toxic side effects. [Summary of the Invention]

[0005] Therefore, in a first aspect of the present invention, a gene construct is provided comprising a first coding sequence encoding a TLR agonist and a second coding sequence encoding a cytokine.

[0006] The inventors have created a novel cancer-killing virus that is remarkably effective in treating BCG non-responder models (and ultimately patients). Advantageously, as shown in Figures 2 and 3, this virus retains potent tumor cytotoxicity with enhanced immunogenicity by incorporating a TLR agonist (e.g., a novel bacterial protein) along with cytokines (e.g., IL-15) that promote NK cell proliferation and CD8 T cell cytotoxicity.

[0007] Preferably, the gene construct includes an expression cassette, one embodiment of which is shown in Figure 1.

[0008] Therefore, a second aspect of the present invention provides an expression cassette comprising the gene construct of the first aspect.

[0009] As can be seen in Figure 1, the construct includes a first nucleotide sequence encoding a TLR agonist (e.g., a novel bacterial protein) and a second nucleotide sequence encoding a cytokine (e.g., IL-15). However, it will be understood that other TLR agonists and cytokines may be used, as will be discussed herein.

[0010] In one embodiment, the TLR agonist may be a bacterial TLR agonist, or it may be a synthetic TLR agonist.

[0011] Preferably, the TLR agonist is a TLR2 agonist, a TLR4 agonist, or a TLR5 agonist. More preferably, the TLR agonist is a TLR2 agonist or a TLR5 agonist.

[0012] TLR agonists can be lipoproteins or lipopeptides. An example of a TLR agonist can be found in Kaczanowska S, Joseph AM, and Davida E. TLR agonists: our best frenemy in cancer immunotherapy. J Leukoc Biol. 2013 Jun;93(6):847-63. doi:10.1189 / jlb.1012501. Epub 2013 Mar 8. PMID:23475577;PMCID:PMC3656332.

[0013] In one preferred embodiment, the TLR2 agonist may be selected from the group consisting of LpqH, LprA, LprG, LpqT, PhoS1, MPB83, MPT83, LAM, AraLAM, LM, PIM2 / 6, TDM, HSP70, MymA, PE_PGRS33, EsxL, PPE18, PPE26, PPE32, PPE57, Lrp, GPLs, PILAM, PPE60, OmpA, and OmpC.

[0014] Preferably, the TLR2 agonist is PPE60 derived from Mycobacterium tuberculosis. In another preferred embodiment, the TLR2 agonist is the outer membrane protein OmpA and / or OmpC derived from Shigella dysenteriae.

[0015] In one preferred embodiment, the TLR4 agonist is FimH. FimH, which is the TLR4 agonist, may be derived from E. coli and / or Salmonella enterica serovar Typhimurium.

[0016] In one preferred embodiment, the TLR5 agonist may be selected from the group consisting of FliC, CagL, CagY, FllA, and FlaB. CagL and CagY may be derived from Helicobacter pylori. FllA may be derived from E. coli. FlaB may be derived from Vibrio vulnificus.

[0017] In one preferred embodiment, the TLR5 agonist is FliC. The TLR5 agonist FliC may be derived from E. coli and / or Burkholderia pseudomallei (BP). Alternatively, the TLR5 agonist FliC may be derived from other Burkholderia species or Salmonella strains, including, but not limited to, Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella gallinarum, Salmonella pullorum, and Salmonella enterica, as well as their subspecies and serotypes.

[0018] As described in the examples, when screening TLR ligands, the inventors identified the bacterial flagellin protein FliC_BP as a particularly effective candidate. In particular, the inventors identified that FliC_BP can induce TLR5 and TLR2-mediated signaling pathways, suggesting either the unique properties of FliC_BP binding to the TLR2 receptor or the occurrence of crosstalk between TLR5 (the primary receptor for flagellin) and TLR2. Thus, this TLR5 / TLR2 ligand offers an opportunity to enhance the antitumor immune response seen in oncolytic viruses through an innate bacterial response.

[0019] Therefore, most preferably, the TLR agonist is a TLR5 agonist.

[0020] Most preferably, the TLR5 agonist is FliC_BP.

[0021] FliC_BP is a bacterial flagellin protein derived from Burkholderia pseudomallei. FliC_BP (GenBank: ABA48561.1) may contain the amino acid sequence of Sequence ID No. 1 provided herein, as follows: MVYRFSARAIRVHARSRRLAGRRTARRSHARFVRIQRWLTERSTQKIRAESRPAPSSFFGVRRNPAQGGRQRTSASARANSGIDGLIQLNLEIFMLGINSNINSLVAQQNLNGSQGALSQAI TRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLTNSLQRIRQLAVQASNGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQVGAN VGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVAIDSSGAAWSSGSTGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATDQANATA MVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETANLSRAQVLQQAGISVLAQANSLPQQVLKLLQ [SEQ ID NO: 1].

[0022] Therefore, in a preferred embodiment, FliC_BP comprises or comprises substantially the amino acid sequence shown in SEQ ID NO: 1, or a fragment or variant thereof.

[0023] In one embodiment, FliC_BP may be encoded by the nucleic acid sequence of Sequence ID No. 2 provided herein, as follows: ATGGTTTACCGCTTTTCCGCGCGGGCGATCCGTGTCCACGCCCGTTCGCGCCGCCTTGCGGGCCGCCGGA CGGCGCGCCGTTCGCATGCGCGATTCGTGCGCATCCAACGTTGGCTCACCGAACGATCGACACAAAAAAT TCGGGCCGAATCTCGCCCTGCCCCCTCAAGTTTTTTTGGCGTACGCCGAAATCCTGCACAAGGCGGCCGG CAAAGAACGAGTGCTTCCGCCCGCGCCAATAGTGGCATTGATGGGCTTATACAGCTCAATTTGGAGATTT TCATGCTCGGAATCAACAGCAACATTAACTCGTTGGTCGCTCAACAGAACCTCAACGGCTCGCAAGGCGC CCTGTCCCAAGCGATCACCCGCCTGTCGTCGGGCAAGCGCATCAACAGCGCGGCGGACGATGCGGCCGGC CTCGCGATCGCCACCCGGATGCAAACGCAGATCAACGGCCTGAACCAGGGCGTGTCGAACGCGAACGACG GCGTGTCGATCCTGCAAACGGCATCGAGCGGCCTGACCTCGCTCACCAACAGCCTGCAGCGTATCCGCCA GCTCGCCGTGCAGGCCTCGAACGGCCCGCTGAGCGCGAGCGACGCGTCGGCGCTGCAACAGGAAGTCGCG CAGCAGATCTCGGAAGTGAACCGTATCGCTTCGCAGACGAACTACAACGGCAAGAACATCCTCGACGGCT CGGCAGGCACGCTGAGCTTCCAGGTCGGCGCGAACGTCGGCCAGACGGTCTCCGTCGACCTCACGCAAAG CATGTCGGCGGCGAAGATCGGCGGCGGCATGGTTCAGACGGGCCAGACGCTCGGCACGATCAAGGTGGCG ATCGACTCGAGCGGCGCGGCCTGGTCGTCGGGCAGCACCGGCCAGGAGACGACGCAGATCAACGTCGTGT CGGACGGCAAGGGCGGCTTCACGTTCACCGATCAGAACAACCAGGCGCTGTCGTCGACGGCCGTGACCGC CGTGTTCGGCTCGTCGACCGCCGGCACGGGCACGGCGGCCTCGCCGTCGTTCCAGACGCTGGCGCTGTCG ACTTCGGCAACCAGCGCGCTGTCCGCGACGGACCAGGCGAACGCCACGGCGATGGTTGCGCAGATCAACG CGGTCAACAAGCCGCAAACGGTCTCGAACCTCGACATCAGCACGCAGACGGGCGCGTACCAGGCGATGGT ATCGATCGACAACGCACTCGGCGACGGTCAACAATCTGCAGGCAACGCTCGGCGCGGCGCAAAACCGCTTC ACCGCGATCGCGACGACGCAGCAAGCCGGCTCGAACAACCTCGCGCAGGCGCAATCGCAAATCCAGAGCG CGGACTTTGCTCAGGAAACCGCGAACCTGTCGCGCGCGCAAGTGCTCCAGCAAGCCGGCATCTCGGTGCT CGCGCAAGCGAACTCGCTGCCGCAGCAAGTGCTGAAGCTCCTGCAATAA[Sequence ID 2].

[0024] Therefore, in a preferred embodiment, FliC_BP is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 2, or a fragment or variant thereof.

[0025] When produced by Burkholderia pseudomallei, flagella are typically not glycosylated. Glycosylation of Burkholderia pseudomallei flagellar sequences may inhibit TLR5 ligand function in mammalian viral systems. To identify N-glycosylation sites in Burkholderia pseudomallei flagella, the inventors ran the sequences through Net N Glyc server website software. Three N-glycosylation sites were found (AA numbers 18, 270, and 358). To avoid N-glycosylation sites, asparagine (N) on the glycosylation sites was replaced with glutamine (Q).

[0026] Therefore, in a preferred embodiment, FliC_BP may include the optimized amino acid sequence of SEQ ID NO: 3 provided herein, as follows: LGINSNINSLVAQQNLQGSQGALSQAITRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLTNSLQRIRQLAVQAS NGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQVGANVGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVAIDSSGAAWSSGS TGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATDQAQATAMVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETAQLSRAQVLQQAGISVLAQANSLPQQVLKLLQ [SEQ ID NO: 3].

[0027] Therefore, in a preferred embodiment, FliC_BP comprises or comprises the optimized amino acid sequence substantially shown in SEQ ID NO: 3, or a fragment or variant thereof.

[0028] In one embodiment, FliC_BP may be encoded by the optimized nucleic acid sequence of Sequence ID No. 4 provided herein, as follows:

[0029] Therefore, in a preferred embodiment, FliC_BP is encoded by the optimized nucleotide sequence, or a fragment or variant thereof, substantially shown in Sequence ID No. 4.

[0030] To induce extracellular secretion of a TLR agonist (e.g., FliC_BP flagellar protein), the inventors further added a CD33 secretion signal. Therefore, in a preferred embodiment, the first coding sequence of the gene construct according to the first embodiment encodes the CD33 secretion signal. Preferably, the CD33 secretion signal is located at the 5' end of the sequence encoding the TLR agonist (e.g., FliC_BP).

[0031] The CD33 secretion signal may include the amino acid sequence of SEQ ID NO: 5 provided herein, as follows: MPLLLLLPLLWAGALA[Sequence ID 5].

[0032] Therefore, in a preferred embodiment, the CD33 secretion signal comprises or consists of the amino acid sequence substantially shown in SEQ ID NO: 5, or a fragment or variant thereof.

[0033] In one embodiment, the CD33 secretion signal may be encoded by the nucleic acid sequence of SEQ ID NO: 6 provided herein, as follows: atg cct ctg ctg ctg ctg ctg cct ctg ctg tgg gct ggg gct ctg gcc [SEQ ID NO: 6].

[0034] Therefore, in a preferred embodiment, the CD33 secretion signal is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 6, or a fragment or variant thereof.

[0035] Therefore, it will be understood that the FliC_BP and CD33 secretion signals may together include the amino acid sequence of SEQ ID NO: 7 provided herein, as follows: MPLLLLLPLLWAGALALGINSNINSLVAQQNLQGSQGALSQAITRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLTNS LQRIRQLAVQASNGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQVGANVGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVAIDSS GAAWSSGSTGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATDQAQATAMVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETAQLSRAQVLQQAGISVLAQANSLPQQVLKLLQ[Sequence ID 7].

[0036] Therefore, in a preferred embodiment, the FliC_BP and CD33 secretion signals include, or consist of, the amino acid sequence substantially shown in SEQ ID NO: 7, or a fragment or variant thereof.

[0037] In one embodiment, the FliC_BP and CD33 secretion signals may be encoded by the nucleic acid sequence of SEQ ID NO: 8 provided herein, as follows:

[0038] Therefore, in a preferred embodiment, the FliC_BP and CD33 secretion signals are encoded by the nucleotide sequence substantially shown in SEQ ID NO: 8, or a fragment or variant thereof.

[0039] Cytokines can be chemokines. Chemokines can be selected from the group consisting of CXCL10, CXCL9, CXCL11, CXCL8, CXCL12, CCL2, CCL3, and CCL5.

[0040] In a preferred embodiment, the cytokine is selected from the group consisting of IL-2, IL-6, IL-10, IL-12, non-secretorized IL-12, IL-15, IL-17, IL-21, IL-33, and type I and type II interferons (IFN-α, IFN-β, IFN-γ).

[0041] However, most preferably, the cytokine is IL-15.

[0042] IL-15 may include the amino acid sequence of SEQ ID NO: 9 provided herein, as follows: MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEKKNIKEFLQSFVHIVQMFINTS[Sequence ID 9].

[0043] Therefore, in a preferred embodiment, IL-15 comprises or consists of the amino acid sequence substantially shown in SEQ ID NO: 9, or a fragment or variant thereof.

[0044] In one embodiment, IL-15 may be encoded by the nucleic acid sequence of Sequence ID No. 10 provided herein, as follows: ATGGACTGGACCTGGATTCTGTTCCTGGTGCAGCAGCAACAAGGGTGCACAGCAACTGGGTGAATGTGATCTCCGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACAGAGTCCGATGTGCACCCCTCTTGCAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGTCA TCTCTCTGGAGAGCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAATTCTCTGAGCTCCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGAAGAAGAACATCAAGGAGTTCCTGCAGTCCTTTGTGCACATCGTGCAGATGTTCATCAATACATCT [SEQ ID NO: 10].

[0045] Therefore, in a preferred embodiment, IL-15 is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 10, or a fragment or variant thereof.

[0046] Preferably, the first coding sequence encoding the TLR agonist and the second coding sequence encoding the cytokine are under the control of one or more promoters. Most preferably, the first coding sequence encoding the TLR agonist is under the control of a first promoter, and the second coding sequence encoding the cytokine is under the control of a second promoter.

[0047] Alternatively, in another embodiment, the gene encoding the TLR agonist and the gene encoding the cytokine are under the control of a single promoter. Therefore, in this embodiment, the gene construct according to the first embodiment includes a first promoter operably linked to a first coding sequence encoding the TLR agonist and a second coding sequence encoding the cytokine. Preferably, under the control of a single promoter, the first and second coding sequences are expressed as a fusion protein. The first and second coding sequences can be separated by a nucleotide sequence encoding a fusion protein linker.

[0048] In a preferred embodiment, a gene construct according to the first embodiment includes a first promoter operably ligated to a first coding sequence encoding a TLR agonist, and / or a second promoter operably ligated to a second coding sequence encoding a cytokine.

[0049] Each of these promoters may be any nucleotide sequence capable of binding to a coding sequence and inducing RNA polymerase to transcribe it. Each of these promoters may be a constitutive or regulatory promoter. Examples of constitutive promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the RSV promoter, and the T7 polymerase promoter.

[0050] Therefore, in a preferred embodiment, this or each promoter may be selected from the group consisting of CMV, RSV, T7 polymerase, EF1a, SV40, PGK1, Ac5, UBI, MP-84, and MP-135. Advantageously, because MP-84 and MP-135, which are adeno-associated virus micropromoters, are small in size, they allow more space for recombinant genes in smaller viruses.

[0051] In a preferred embodiment, the first promoter operably ligated to a first coding sequence encoding a TLR agonist (e.g., FliC_BP) is a cytomegalovirus (CMV) promoter. One embodiment of the nucleotide sequence encoding the cytomegalovirus (CMV) promoter is referenced herein as Sequence ID No. 12: [Sequence ID 12].

[0052] Therefore, preferably, the first promoter comprises or consists of the nucleic acid sequence substantially represented in SEQ ID NO: 12, or a fragment or variant thereof.

[0053] Preferably, the second promoter operably ligated to a second coding sequence encoding a cytokine is an RSV promoter. One embodiment of the nucleotide sequence encoding the RSV promoter is referenced herein as Sequence ID No. 13: aatgtagtctttgcaatacacttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga aggcaacagacaggtctgacatggattggacgaaccactgaattccgcattgcagagataattgtatttaagtgcctagctcgatacaataaacgccatttgaccattcaccacattggtgtgcacc [SEQ ID NO: 13].

[0054] Therefore, preferably, the second promoter comprises or consists of the nucleic acid sequence substantially represented in SEQ ID NO: 13, or a fragment or variant thereof.

[0055] As can be seen in Figure 1, the gene construct (or expression cassette) contains two nucleotide sequences that encode two poly(A) tails.

[0056] Preferably, the gene construct or expression cassette includes a nucleotide sequence encoding a poly-A tail. More preferably, the gene construct or expression cassette includes first and second nucleotide sequences encoding first and second poly-A tails, respectively. Preferably, the first and second nucleotide sequences encoding poly-A tails are adjacent to the cassette.

[0057] Preferably, the first poly-A tail code sequence is located 3' to the first code sequence that codes for the TLR agonist (e.g., FliC_BP).

[0058] Preferably, the second poly-A tail coding sequence is located 3' to the second coding sequence encoding a cytokine (e.g., IL-15).

[0059] One embodiment of a nucleotide sequence encoding a first and / or second poly-A tail (BGH poly-A tail for CMV) is referenced herein as Sequence ID No. 14: ccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatg caatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacag [SEQ ID NO: 14].

[0060] Therefore, preferably, the first and / or second poly-A tail comprises a nucleic acid sequence substantially represented in SEQ ID NO: 14, or a fragment or variant thereof.

[0061] One embodiment of a nucleotide sequence encoding a first and / or second poly-A tail (SV40 poly-A tail for RSV) is referenced herein as Sequence ID No. 15, as follows: aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgc[Sequence code 15].

[0062] Therefore, preferably, the first and / or second poly-A tail comprises a nucleic acid sequence substantially represented in SEQ ID NO: 15, or a fragment or variant thereof.

[0063] Preferably, the gene construct of the first embodiment or the expression cassette of the second embodiment is retained in the oncolytic virus.

[0064] Accordingly, a third aspect of the present invention provides an oncolytic virus comprising a gene construct of the first aspect or an expression cassette of the second aspect.

[0065] As can be seen in Figure 1, the oncolytic virus contains a first nucleotide sequence encoding a TLR agonist (e.g., a novel bacterial protein, preferably FliC_BP) under the control of a first promoter (e.g., CMV), and a second nucleotide sequence encoding a cytokine (e.g., IL-15) under the control of a second promoter (e.g., RSV). The virus also contains two adjacent poly-A tails on both sides of the expression cassette.

[0066] The inventors have found FliC_BP to be a particularly effective candidate for enhancing the antitumor immune response observed in oncolytic viruses through an innate bacterial response. In particular, the inventors discovered that FLiC_BP can induce TLR5 and TLR2-mediated signaling pathways, suggesting either the unique properties of FliC_BP that bind to the TLR2 receptor or the occurrence of crosstalk between TLR5 (the primary receptor for flagellin) and TLR2. This crosstalk between TLR2 and TLR5 is likely to amplify the production of inflammatory cytokines, thereby contributing to a more robust and immediate immune response. The inventors believe they have incorporated an expression cassette encoding FliC_BP into an oncolytic virus for the first time.

[0067] Therefore, a fourth aspect of the present invention provides an oncolytic virus comprising a nucleotide sequence encoding Burkholderia pseudomallei flagellin protein (FliC_BP).

[0068] The nucleotide sequence encoding FliC_BP may be as described above in relation to the first or second embodiment. For example, the nucleotide sequence encoding FliC_BP may include, or be derived from, the nucleotide sequence substantially shown in SEQ ID NO: 2 or 4, or a fragment or variant thereof.

[0069] Preferably, the oncolytic virus according to the fourth embodiment includes a first promoter operably ligated to a nucleotide sequence encoding FliC_BP.

[0070] The promoter may be as described above in relation to the first or second embodiment. For example, the promoter may be selected from the cytomegalovirus (CMV) promoter, the RSV promoter, and the T7 polymerase promoter. In a preferred embodiment, the promoter operably ligated to the nucleotide sequence encoding FliC_BP is the cytomegalovirus (CMV) promoter, and preferably comprises or consists of the nucleic acid sequence substantially shown in SEQ ID NO: 12, or a fragment or variant thereof.

[0071] As used herein, the term oncolytic virus, in relation to the third or fourth aspect, is understood to be a virus that infects and lyses cancer cells but does not infect or lyse normal or healthy cells. Oncolytic viruses may be naturally occurring viruses that are oncolytic, or they may be engineered by modifying one or more viral genes to enhance tumor selectivity.

[0072] Preferably, the oncolytic virus is selected from the group consisting of herpes simplex virus (HSV), coxsackievirus, maraba virus, measles virus (MV), Newcastle disease virus (NDV), poliovirus, reovirus, retrovirus, Seneca Valley virus (SVV), alphavirus, such as Semryqui forest virus (SFV) and Sindbisvirus (SINV), varicella stomatitis virus (VSV), Sindbisvirus (SBV), adenovirus, poxvirus, parvovirus, flavivirus, such as Zika virus, paramyxovirus, picornavirus, and rhabdovirus.

[0073] Most preferably, the oncolytic virus is herpes simplex virus (HSV). The HSV virus may be HSV-1 or HSV-2. Preferably, the HSV virus is HSV-1. Most preferably, the HSV virus is HSV-1 strain 17+.

[0074] The inventors have discovered that oncolytic viruses (e.g., HSV) contribute to therapeutic effects when in a living, replicated state, resulting in maximum oncolysis of bladder tumor cells. Advantageously, living, replicated oncolytic viruses facilitate the direct destruction of cancer cells and promote the release of therapeutic proteins such as IL-15 and FliC_BP along with tumor antigens. This release is essential for stimulating both innate and adaptive immune responses to tumor antigens and viral proteins, resulting in sustained anti-tumor immunity.

[0075] Therefore, in some embodiments, the oncolytic virus is a live replicating virus. In some embodiments, the oncolytic virus is a live replicating HSV. In some embodiments, the oncolytic virus is a live replicating HSV-1 or HSV-2.

[0076] The HSV-1 neurotoxic protein, infection cell protein 34.5 (ICP34.5), is necessary for HSV-1 infection of neurons and other healthy cells because it binds to and blocks the PKR pathway, enabling viral replication. ICP34.5 blocks the activation of the PKR pathway by binding to and activating PP1α phosphatase, which in turn prevents the arrest of protein translation by eIF-2 by dephosphorylating eIF-2α. Therefore, for cancer cell infection, the absence of the ICP34.5 gene is preferable.

[0077] The HSV-1 genome is a single linear double-stranded DNA molecule approximately 152,000 bp long. It is divided into two distinct segments called the long (UL) and short (US) segments. Short regions of repetitive sequences occur at the genome ends and between the L and S segments. ICP34.5 is encoded within two short regions of repetitive sequences in the UL segment. Therefore, there are two copies of the gene per genome. The first ICP34.5 gene is encoded between 513–1539 bp in the genome, and the second ICP34.5 gene is encoded between 124832–125858 bp.

[0078] Therefore, in a preferred embodiment, the oncolytic virus contains a functionally deleted ICP34.5 gene. The ICP34.5 gene may be functionally deleted if it is absent, destroyed, or non-functional. More preferably, the oncolytic herpes simplex virus contains a functional deletion of at least two copies of the ICP34.5 gene.

[0079] Advantageously, this functional deletion affects the elimination of HSV-1 pathogenicity and also enhances viral replication in cancer cells. Infection of healthy cells with ICP34.5-deficient HSV-1 activates PKR, resulting in sterile infection. In contrast, this HSV can replicate in cancer cells because PKR activity is not activated.

[0080] Functional deletion of the ICP34.5 gene can be achieved by introducing one or more mutations into the viral DNA such that the expressed protein is non-functional or has low activity. Alternatively, functional deletion of the ICP34.5 gene can be achieved by exposing the virus to an inhibitor of the encoded protein, such as ICP34.5-specific RNA, such as siRNA.

[0081] However, preferably, a functional deletion of the ICP34.5 gene is achieved by incorporating a gene construct according to the first embodiment or an expression cassette according to the second embodiment into the ICP34.5 gene.

[0082] Preferably, the oncolytic virus contains a functionally deleted ICP47 gene. The ICP47 gene may be functionally deleted if it is absent, destroyed, or non-functional.

[0083] This functional deletion of ICP47 has multiple effects, including improving the lytic activity of HSV-1 ICP34.5 / - mutants. US11 encodes UL11, which binds to PKR, inhibiting the phosphorylation of eIF-2α and thereby enabling HSV-1 replication. Furthermore, US11 is normally expressed late in infection, well after ICP34.5 inhibits PKR. However, such deletions in HSV viruses result in regulation of US11 by the α47 promoter, causing UL11 to be expressed as a pre-early gene, which blocks PKR activity before PKR can terminate protein synthesis. In many cancer cells, the type I IFN signaling pathway is disrupted, and in the absence of ICP34.5, viral replication occurs in cancer cells lacking normal IFN signaling, while normal cells are less tolerant because IFN signaling is not impaired. This feature of the HSV-1 ICP34.5 / - ICP47- mutant adds an important safety component, as it disrupts infection of normal cells in the absence of PKR and type I IFN signaling defects.

[0084] Therefore, in a preferred embodiment, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' coding sequence encoding a cytokine (e.g., IL-15) and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' coding sequence encoding a TLR agonist (e.g., FliC_BP) and a 3' coding sequence encoding a cytokine (e.g., IL-15). The use of 5' and 3' is not intended to indicate that the feature is either upstream or downstream, but rather that the feature is not necessarily a terminal feature.

[0085] In certain embodiments, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' coding sequence encoding a cytokine (e.g., IL-15), a first promoter (e.g., RSV), a second promoter (e.g., CMV), and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' coding sequence encoding a TLR agonist (e.g., FliC_BP), a first promoter (e.g., CMV), a second promoter (e.g., RSV), and a 3' coding sequence encoding a cytokine (e.g., IL-15).

[0086] Alternatively, in another embodiment, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' first promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), a second promoter (e.g., CMV), and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' first promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), a second promoter (e.g., RSV), and a 3' coding sequence encoding a cytokine (e.g., IL-15).

[0087] In certain embodiments, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' poly-A tail, a coding sequence encoding a cytokine (e.g., IL-15), a first promoter (e.g., RSV), a second promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail. Alternatively, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' poly-A tail, a coding sequence encoding a TLR agonist (e.g., FliC_BP), a first promoter (e.g., CMV), a second promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), and a 3' poly-A tail.

[0088] Alternatively, in another particular embodiment, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' first promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), a poly-A tail, a second promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail. Alternatively, an oncolytic virus according to the third embodiment may include, in this particular order, a 5' first promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), a poly-A tail, a second promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), and a 3' poly-A tail.

[0089] In a preferred embodiment, the oncolytic virus according to the fourth aspect may include, in this particular order, a 3' coding sequence encoding a 5' promoter (e.g., CMV) and a TLR agonist (e.g., FliC_BP).

[0090] In certain embodiments, the oncolytic virus according to the fourth aspect may include, in this particular order, a coding sequence encoding a 5' promoter (e.g., CMV), a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail.

[0091] The inventors have demonstrated that a combination of TLR agonists and cytokines in the context of oncolytic viruses (e.g., HSV) enhances the oncolytic activity of the virus while simultaneously producing enhanced acquired immunity against tumors. Therefore, the inventors have surprisingly discovered that antigens are not necessary to create therapeutic agents for cancer.

[0092] Therefore, in some embodiments, the gene constructs, expression cassettes, and oncolytic viruses according to the present invention do not contain antigens (e.g., tumor antigens or cancer antigens).

[0093] From the foregoing, those skilled in the art will understand the nucleotide and amino acid sequences of the oncolytic virus embodiments of the third and fourth embodiments.

[0094] In some embodiments, a shuttle vector may be used to produce an oncolytic virus according to the present invention. Therefore, to avoid misunderstanding, the nucleotide sequence encoding the shuttle vector used to produce an oncolytic virus according to the present invention (known as p-34.5 DC RSV CMV) is provided herein as SEQ ID NO: 11:

[0095] Alternatively, in another embodiment, a nucleotide sequence encoding a shuttle vector (known as p-34.5 DC CMV S-FLiC-M3 RSV) used to produce an oncolytic virus according to the present invention is provided herein as Sequence ID No. 16:

[0096] Alternatively, in another embodiment, a nucleotide sequence encoding a shuttle vector (known as p-34.5 DC CMV RSV IL-15ED) used to produce an oncolytic virus according to the present invention is provided herein as Sequence ID No. 17:

[0097] Alternatively, in another embodiment, a nucleotide sequence encoding a shuttle vector (known as p-34.5 DC RSV CMV S-FLiC-M3) used to produce an oncolytic virus according to the present invention is provided herein as Sequence ID No. 18:

[0098] Therefore, in a preferred embodiment, the shuttle vector used to produce the oncolytic virus according to the present invention is encoded by the nucleotide sequence shown in SEQ ID NOs: 11, 16, 17, or 18, or a fragment or variant thereof.

[0099] Oncolytic viruses according to third and fourth aspects of the present invention are particularly suitable for the treatment of cancer, preferably bladder cancer.

[0100] Accordingly, according to a fifth aspect of the present invention, a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect is provided for therapeutic use.

[0101] According to the sixth aspect, a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect is provided for use in the treatment, prevention, or improvement of cancer.

[0102] According to the seventh aspect, a method is provided for treating, preventing or improving cancer in a subject, comprising administering, or having administered to, a therapeutically effective amount of a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect to a subject in need of such treatment.

[0103] The cancer could be bladder cancer.

[0104] In a preferred embodiment, the cancer is bladder cancer. More preferably, the cancer is nonmuscle-invasive bladder cancer (NMIBC). Preferably, the bladder cancer is BCG-refractory bladder cancer.

[0105] Advantageously, the gene constructs, expression cassettes, and oncolytic viruses according to the present invention facilitate the direct destruction of cancer cells and promote the release of therapeutic proteins such as IL-15 and FliC_BP along with tumor antigens. This release is essential for stimulating both innate and adaptive immune responses to tumor antigens and viral proteins, resulting in sustained anti-tumor immunity.

[0106] Therefore, in some embodiments, the gene constructs, expression cassettes, and oncolytic viruses according to the present invention stimulate innate and adaptive immune responses.

[0107] In some embodiments, gene constructs, expression cassettes, and oncolytic viruses according to the present invention stimulate TLR2 and TLR-5 mediated signaling pathways.

[0108] It will be understood that oncolytic viruses according to the present invention may be used in pharmaceuticals that can be used as monotherapy (i.e., use of oncolytic viruses alone) for the treatment of cancer. Alternatively, oncolytic viruses according to the present invention may be used as an adjunct to or in combination with known therapies for treating, improving, or preventing cancer.

[0109] The oncolytic virus of the present invention can be combined in compositions having many different forms, particularly depending on how the composition is used. For example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, polyplex, emulsion, lipid nanoparticles (e.g., containing peptides, DNA, or RNA on or encapsulated on a surface), or any other suitable form that can be administered to humans or animals requiring vaccination. Lipid nanoparticles may comprise one or more components selected from the group consisting of cationic lipids (preferably ionizable); phosphatidylcholine; cholesterol; and polyethylene glycol (PEG)-lipids. It will be understood that the vehicle of the pharmaceutical according to the present invention should exhibit good tolerability in the subject to which it is administered.

[0110] The pharmaceuticals containing the oncolytic virus of the present invention can be used in many ways. For example, oral administration may be required, in which case the drug may be contained in a composition that can be taken orally, for example, in the form of tablets, capsules, or liquids. The drugs and compositions containing the pharmaceuticals of the present invention can be administered by inhalation (e.g., intranasally). The compositions can also be formulated for topical use. For example, a cream or ointment may be applied to the skin.

[0111] Furthermore, the oncolytic virus of the present invention may be incorporated into a sustained-release or delayed-release device. Such a device can be inserted, for example, on or under the skin, and the drug can be released over several weeks or even months. The device can be positioned at least adjacent to the treatment site. Such a device may be particularly advantageous when long-term treatment with the oncolytic virus is required and usually necessitates frequent administration (e.g., at least daily injections).

[0112] However, in preferred embodiments, pharmaceuticals according to the present invention may be administered to a subject by injection into the bloodstream, muscle, skin, or directly to a site requiring treatment. The injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), or intramuscular (bolus or infusion). In some embodiments, the injection is intravenous. Typically, the injection is intravesical or intratumor. In the case of intravesical injection, these may be administered into the bladder by catheter.

[0113] The required amount of oncolytic virus is determined by its biological activity and bioavailability, which will be understood to depend further on the mode of administration, the physiochemical properties of the oncolytic virus, and whether it is used as monotherapy or in combination therapy. The frequency of administration is also influenced by the half-life of the activator in the subject being treated. The optimal dose to be administered can be determined by those skilled in the art and will vary depending on the specific oncolytic virus used, the strength of the composition, the mode of administration, and the type and stage of cancer. Additional factors specific to the subject being treated, including age, weight, sex, diet, and time of administration, may necessitate adjustments to the dose.

[0114] Generally, depending on the drug used, a daily dose of the oncolytic virus of the present invention between 0.001 μg / kg body weight and 100 mg / kg body weight may be used for immunization. More preferably, the daily dose of the drug is between 1 μg / kg body weight and 100 mg / kg body weight, more preferably between 10 μg / kg and 10 mg / kg body weight, and most preferably between about 100 μg / kg and 10 mg / kg body weight. In some embodiments, a dose of the oncolytic virus of the present invention between 10E4 plaque-forming units (PFUs) / mL and 10E9 PFUs / mL may be used for immunization.

[0115] The daily dose may be administered as a single dose (e.g., once daily injection). Alternatively, the oncolytic virus may require two or more doses during the day. For example, the oncolytic virus may be administered as an initial primer and subsequent boost, or as two boosts administered at intervals of one week or one month. Preferably, the oncolytic virus may be administered as an initial primer and subsequent boost, administered at intervals of two to six weeks. Using known procedures such as those conventionally used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), specific formulations of the oncolytic virus according to the present invention and precise treatment regimens (e.g., daily dose and frequency of administration of the drug) can be formed.

[0116] The inventors believe they have proposed for the first time an oncolytic virus containing a gene encoding a TLR agonist and a gene encoding a cytokine, or an oncolytic virus containing a gene encoding FliC_BP.

[0117] According to an eighth aspect of the present invention, a pharmaceutical composition is provided comprising a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect, and a pharmaceutically acceptable vehicle.

[0118] In a ninth aspect, the present invention also provides a process for producing a pharmaceutical composition according to an eighth aspect, comprising combining a therapeutically effective amount of a gene construct according to a first aspect, an expression cassette according to a second aspect, or an oncolytic virus according to a third or fourth aspect with a pharmaceutically acceptable vehicle.

[0119] The "subject" may be a vertebrate, mammal, or domesticated animal. Therefore, compositions and pharmaceuticals according to the present invention may be used to treat any mammal, e.g., livestock (e.g., horses), pets, or for other veterinary purposes. However, most preferably, the subject is human.

[0120] The "therapeutic dose" of oncolytic virus is any amount, preferably prophylactic, that is required to improve, prevent or treat any given disease when administered to a subject.

[0121] For example, the oncolytic virus of the present invention can be used in amounts of about 0.001 μg to about 1 mg, preferably about 0.001 μg to about 500 μg. The amount of oncolytic virus is preferably about 0.01 μg to about 250 μg, and most preferably about 0.1 μg to about 100 μg. Preferably, the oncolytic virus according to the present invention is administered in doses of 1 to 50 μg.

[0122] The oncolytic virus of the present invention may further comprise a pharmaceutically acceptable vehicle. Where used herein, “pharmaceutically acceptable vehicle” is any known compound or combination of known compounds that are known to those skilled in the art as useful for formulating pharmaceutical compositions.

[0123] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or a tablet. A solid pharmaceutically acceptable vehicle may contain one or more substances that can also act as a flavoring agent, lubricant, solubilizer, suspending agent, dye, filler, fluidizer, compression aid, inert binder, sweetener, preservative, dye, coating agent, or tablet disintegrant. The vehicle may also be an encapsulating material. In the powder, the vehicle is a finely divided solid that is a mixture with a finely divided activator according to the present invention. In the tablet, the activator (e.g., an oncolytic virus according to the present invention) can be mixed in an appropriate proportion with a vehicle having the required compression properties and compressed to the desired shape and size. The powder and tablet preferably contain up to 99% activator. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting-point waxes, and ion-exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or the like. In yet another embodiment, the oncolytic virus or pharmaceutical composition may be lyophilized.

[0124] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in the preparation of liquids, suspensions, emulsions, syrups, elixirs, and pressurized compositions. Oncolytic viruses according to the present invention may be dissolved or suspended in water, organic solvents, a mixture of both, or pharmaceutically acceptable liquid vehicles such as pharmaceutically acceptable oils or fats. Liquid vehicles may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful for compositions in sterile liquid form for parenteral administration. Liquid vehicles for pressurized compositions may be halogenated hydrocarbons or other pharmaceutically acceptable propellants.

[0125] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered, for example, by subcutaneous, intradermal, intrathecal, epidural, intraperitoneal, intravenous, and especially intramuscular injection, or by urinary catheterization via a urethral catheter. The nucleic acid sequences or expression cassettes of the present invention can be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injection media.

[0126] The oncolytic virus of the present invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., saline or glucose sufficient to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monolaurate, polysorbate 80 (sorbitol copolymerized with ethylene oxide and its anhydride oleate ester), etc. The oncolytic virus according to the present invention may also be administered orally in the form of either a liquid or solid composition. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. The pharmaceutical compositions may be frozen, freeze-dried, or lyophilized.

[0127] It will be understood that the present invention extends to any nucleic acid or peptide, or variants, derivatives or analogs thereof, comprising substantially amino acid or nucleic acid sequences of any of the sequences referred herein, including variants or fragments thereof. The terms “substantially amino acid / nucleotide / peptide sequences,” “variant,” and “fragment” may be sequences having at least 40% sequence identity with any one of the amino acid / nucleotide / peptide sequences referred herein, for example, sequences having 40% identity with sequences identified as SEQ ID NOs. 1 to 20, etc.

[0128] Amino acid / polynucleotide / polypeptide sequences having more than 65% sequence identity, more preferably more than 70%, even more preferably more than 75%, and still more preferably more than 80% sequence identity with any of the sequences mentioned herein are also conceivable. Preferably, the amino acid / polynucleotide / polypeptide sequences have at least 85% identity with any of the sequences mentioned herein, more preferably at least 90% identity with any of the sequences mentioned herein, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0129] Those skilled in the art will understand how to calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences, one must first prepare an alignment of the two sequences, and then calculate the sequence identity value. The percentage of identity between two sequences can take different values ​​depending on: (i) the method used to align the sequences, e.g., structural alignment from ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or 3D comparison; and (ii) the parameters used by the alignment method, e.g., local alignment versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional form and constant.

[0130] After alignment, there are many different ways to calculate the percentage of identity between two sequences. For example, the number of identities can be divided by (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage of identity is strongly length-dependent. Thus, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur by chance.

[0131] Thus, it will be understood that the precise alignment of protein or DNA sequences is a complex process. ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882), a common multiple alignment program, is a preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignment, gap opening penalty = 15.0, gap elongation penalty = 6.66, and matrix = identity. For protein alignment, gap opening penalty = 10.0, gap elongation penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment, ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to change these and other parameters for optimal sequence alignment.

[0132] Preferably, the percentage of identity between two amino acid / polynucleotide / polypeptide sequences may then be calculated by an alignment such as (N / T)*100, where N is the number of positions in which the sequences share identical residues, and T is the total number of positions compared, including or excluding gaps and overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage of identity between two sequences includes (i) preparing a sequence alignment using, for example, a ClustalW program with an appropriate set of parameters as described above; and (ii) inserting the values ​​of N and T into the following formula: -Sequence Identity=(N / T)*100.

[0133] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by sequences that hybridize to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3 × sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2 × SSC / 0.1% SDS at about 20–65°C. Alternatively, substantially similar polypeptides may differ from the indicated sequences, e.g., sequences of sequence numbers 1–20, which are amino acid sequences, by at least one, but less than five, ten, twenty, fifty, or 100 amino acids.

[0134] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein it encodes, thereby providing a functional variant thereof. A suitable nucleotide variant is one that has a sequence that is altered by the substitution of a different codon encoding the same amino acid in the sequence, and thus produces a silent (synonymous) change. Other suitable variants are variants that have a homologous nucleotide sequence but are altered by the substitution of a different codon encoding an amino acid having a side chain with similar biophysical properties to the amino acid being substituted, resulting in a conserved change, and include all or part of the sequence. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood that which amino acids may be substituted with those having similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0135] All of the features described herein (including any attached claims, abstracts, and drawings), and / or all of the steps of any method or process disclosed herein, may be combined with any of the above embodiments in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive.

[0136] To better understand the present invention and to illustrate how embodiments of the present invention can be carried out, the accompanying drawings are referred to hereby as examples. [Brief explanation of the drawing]

[0137] [Figure 1] This is a schematic diagram of one embodiment of an oncolytic virus according to the present invention. The oncolytic herpes simplex virus (oHSV-FliC_BP-IL-15) is derived from wild-type (WT) HSV-1 strain 17+ and has two gene deletions, namely, (i) deletions of the genes encoding ICP34.5 and (ii) deletions of the genes encoding ICP47. oHSV-FliC_BP-IL-15 expresses Burkholderia pseudomallei flagellin protein (Genbank ABA48561.1, TLR5 ligand) under the CMV promoter and expresses IL-15 (GenBank:AF031167.1) under the RSV promoter. [Figure 2A] This figure shows the expression of TLR ligands and IL-15 from plasmids and oHSV virus. TLR ligand plasmids or oHSV virus were screened using a TLR receptor / NF-κB reporter assay: a) TLR4 and 5 (Promega); b) TLR2 ligand plasmid (Invivogen); c) oHSV-FliC_BP (Promega). d) Concentration of IL-15 detected using an IL-15 cell bioassay (Promega) in the supernatant from 5637 cells infected with oHSV-IL-15 for 48 hours at MOI 1. e) Partial blockade of the TLR5-MyD88-NF-κB pathway activated by the supernatant from 5637 cells infected with oHSV-FliC_BP for 24 hours at MOI 1 using a TLR5 inhibitor against the soluble ectodomain of human TLR5-TLR5 receptor (α Hu TLR5-Fc, InvivoGen). f) Immunostaining of flagellin bacterial protein (green) derived from BP, performed on 5637 cells infected with oHSV-FliC_BP virus for 24 hours at MOI 1. [Figure 2B]This figure shows the expression of TLR ligands and IL-15 from plasmids and oHSV virus. TLR ligand plasmids or oHSV virus were screened using a TLR receptor / NF-κB reporter assay: g) THP-1 cells (+PMA) were treated with UV-inactivated oHSV or oHSV FLIC BP. THP-1 cells were incubated for 24 hours and qPCR was performed for innate cytokines. [Figure 3] a) Graph of the mean tumor volume of subcutaneous MB49 tumors treated four times over eight days with 108 pfu of oHSV, oHSV-FliC_BP, oHSV-IL-15, or oHSV-FliC_BP-IL-15 (n=10). b) Graph of the Kaplan-Meier survival curve. c) Graph of the rechallenge experiment using the same bladder cancer cell line (MB49) in untreated and cured mice. e-g) Graphs of FACS analysis measuring TNF-α in spherocytes from cured and untreated mice, in co-culture, alone, or with MB49 tumor cells or PMA / ionomycin. e) Graphs of CD4, f) CD8, g) NK. [Figure 4] This figure shows the knockout of ICP47 from the HSV-1 genome. A plasmid (pUC57 del ICP47) containing sequences of two HSV-1 flanking regions on either side of ICP47 was designed and synthesized. The CMV EGPA pA cassette generated by PCR was produced from pcDNA3.1+ EGFP. The PCR product was cleaved with Bbs-1 and then inserted between the flanking regions of ICP47 to produce pUC57 del ICP47 CMV EGFP pA. This viral shuttle vector was linearized (ssp-1) and transfected into BHK cells using calcium phosphate in addition to BHK DNA from HSV-1 infected cells. The resulting homologous recombinants were purified by plaque to obtain clones of HSV-1 17+ 47- EGFP. The CMV EGFP pA cassette was removed from the virus (HSV-1 17+ 47- EGFP) by homologous recombination using pUC57 del ICP47, thereby obtaining a clone of HSV-1 17+ 47-. [Figure 5] This figure shows a plasmid map of p34.5 DC EGFP, which contains sequences of two HSV-1 flanking regions on both sides of ICP34.5. p34.5 DC EGFP contains a dual expression cassette (RSV-pA, CMV pA). Below the CMV promoter is the synthetic green fluorescent gene (EGFP). [Figure 6] This figure shows the plasmid map of p34.5 CMV RSV IL-15ED, which contains the sequences of two ICP34.5 flanking regions and IL-15 ED. p34.5 CMV RSV IL-15ED contains a dual expression cassette (RSV-pA, CMV pA). [Figure 7] This figure shows the plasmid map of p34.5 CMV FLIC BP RSV IL-15ED, which contains the sequences of two ICP34.5 adjacent regions, IL-15 ED and FLIC BP. p34.5 CMV FLIC BP RSV IL-15ED contains a dual expression cassette (RSV-pA, CMV pA). [Figure 8] This figure shows the inactivation of HSV after exposure to 300J of UV light. [Figure 9] This figure shows the survival rates of four TCC bladder cancer cell lines (T24, TCCSUP, Ku19-19, and VMCUB) treated with HSV / HSV5-15, with and without UV inactivation. [Figure 10] This figure shows TLR2 stimulation determined using HEK-Blue® hTLR-2 detection: a) plasmid DNA (containing FLiC BP ligand); and b) FLiC BP ligand expressed from oncolytic HSV (HSV5). [Modes for carrying out the invention] [Examples]

[0138] The inventors designed and tested a novel oncolytic virus for the treatment of nonmuscle-invasive bladder cancer (NMIBC). Considering the issues associated with BCG (i.e., non-responders and / or toxicity), the inventors attempted to identify a single TLR agonist that would provide an alternative therapeutic option to BCG through non-toxic stimulation of NK and NKT cells, which have been shown to play a crucial role in the BCG-induced antitumor response in the treatment of NMIBC. The inventors conducted extensive screening of bacterial TLR ligands with the aim of identifying those suitable for expression in oncolytic herpes simplex virus (oHSV). As a result of the screening, the inventors selected flagellin from Burkholderia pseudomallei, i.e., FliC BP, a TLR5 ligand. In addition to the TLR ligand, the inventors investigated the effects of oHSV, which also encodes the cytokine IL-15. The genes encoding FliC BP and IL15 were retained in HSV strains lacking two genes, namely ICP34.5 and ICP47.

[0139] material and method Production of oncolytic HSV-1 virus All oncolytic viruses constructed and used were based on one strain of human herpes simplex virus 17+ (Genbank X14112) obtained from ECACC.

[0140] HSV-1 ICP47 gene knockout To knock out ICP47 from the HSV-1 genome, a plasmid (pUC57 del ICP47) containing sequences of two HSV-1 flanking regions (Δ47 143671~145261 bp, 145581~146461 bp Genbank X14112) on both sides of ICP47 was designed and synthesized (see Figure 4). The CMV EGFP pA cassette generated by PCR was produced from pcDNA3.1+ EGFP using primers CGFPA F (cloning primer 5) GAGGAAGACGAGGAGACGGGCCAGATATACGCGTTG [SEQ ID NO: 19] and AGCTCCTCGTCTTCCCAATCCTCCCCCTTG [SEQ ID NO: 20]. The PCR product was cleaved with Bbs-1 and then inserted between the flanking regions of ICP47 to construct pUC57 del ICP47 CMV EGFP pA. This viral shuttle vector was linearized (ssp-1) and transfected into BHK cells using calcium phosphate in addition to BHK DNA from HSV-1 infected cells. The resulting homologous recombinants were purified by plaque to obtain clones of HSV-1 17+ 47- EGFP. The CMV EGFP pA cassette was removed from the virus (HSV-1 17+ 47- EGFP) by homologous recombination using pUC57 del ICP47, thereby obtaining clones of HSV-1 17+ 47-.

[0141] Knockout of the HSV-1 ICP34.5 gene To knock out the ICP34.5 gene from HSV-1 17+ 47-, a plasmid (p34.5- DC) was generated containing sequences of two HSV-1 facilitation regions (Δ34.5 123462~124958 bp, 125713~126790 bp Genbank X14112) on both sides of ICP34.5 (see Figure 5). The p34.5- DC contained a dual expression cassette (RSV-pA, CMV pA). Under the CMV promoter, the inventors cloned the synthetic green fluorescent gene (EGFP) Genbank AF188479.1 derived from pcDNA3.1+ EGFP (BamHI, MfeI) to produce p34.5 DC EGFP. This viral shuttle vector was linearized (ssp-1) and transfected into BHK cells using calcium phosphate in addition to BHK DNA from HSV-1 infected cells. The obtained homologous recombinants were purified using plaque analysis to obtain clones of HSV-1 17+ 34.5-EGFP 47-.

[0142] Insertion of recombinant flagellum (Burkholderia pseudomallei Bp) sequence into HSV-1 17+ 34.5- 47- To express Burkholderia pseudomallei-derived flagella (Genbank ABA48561.1, TLR5 ligand) in oncolytic HSV-1 17+ 34.5-47-, a plasmid (pcDNA3.1+S-FLIC BP-M3) was designed and synthesized. Like all bacterial proteins, flagella, when produced by Burkholderia pseudomallei, are typically unglycosylated. Glycosylation of the Bp sequence can inhibit TLR5 ligand function. To identify N-glycosylation sites in the Bp flagella, the sequence was subjected to Net N Glyc server website software. While this software did not identify the N-glycosylation mechanism or the signaling peptides involved, three N-glycosylation sites were found (AA numbers 18, 270, and 358). To avoid N-glycosylation sites, asparagine (N) on the glycosylation sites was replaced with glutamine (Q). To induce extracellular secretion of flagellar protein, a CD33 secretion signal was added. This recombinant sequence was synthesized and then cloned into pcDNA3.1+ (BamH, EcoRI) to obtain pcDNA3.1+S-FLIC BP-M3. To construct a viral shuttle vector, S-FLIC BP-M3 was subcloned from pcDNA3.1+S-FLIC BP-M3 to p34.5- DC under the CMV promoter (BamHI, MfeI) to obtain p34.5-DC S-FLIC-BP-M3. To obtain recombinant virus, p34.5-DC S-FLIC-BP-M3 was linearized (ssp-1), added to BHK DNA from HSV-1 infected cells, and transfected into BHK cells using calcium phosphate. The resulting homologous recombinants were purified by plaque purification to obtain clones of HSV-1 17+ 34.5- S-FLIC BP-M3 47-.

[0143] Insertion of recombinant IL-15 sequence into HSV-1 17+ 34.5-47- To express human interleukin 15 (GenBank:AF031167.1) in oncolytic HSV-1 17+ 34.5-47- cells, a plasmid (pcDNA3.1+IL-15ED) was designed and synthesized (see Figure 6). This recombinant sequence was synthesized and then cloned into pcDNA3.1+ (BamH, EcoRI). To construct a viral shuttle vector containing IL-15ED, the cytokine sequence was subcloned from pcDNA3.1+IL-15ED to the p34.5- DC plasmid under the RSV promoter (PmeI, Xcm-I). To obtain recombinant virus, p34.5- DC IL-15ED was linearized (ssp-1) and transfected into BHK cells using calcium phosphate in addition to BHK DNA from HSV-1 infected cells. The obtained homologous recombinants were purified using plaque analysis to obtain clones of HSV-1 17+ 34.5- IL-15ED 47-.

[0144] Insertion of both recombinant IL-15 and flagella (Burkholderia pseudomallei Bp) sequences into HSV-1 17+ 34.5-47- To express human interleukin 15 and flagellar (Bp) sequences from oncolytic HSV-1 17+ 34.5-47-, a viral shuttle plasmid containing IL-15ED was cleaved with BamHI and MfeI, and the flagellar sequence was inserted under the CMV promoter (see Figure 7). To obtain recombinant viruses, p34.5- DC IL-15ED / S-FLIC-BP-M3 was linearized (ssp-1), added to BHK DNA from HSV-1 infected cells, and transfected into BHK cells using calcium phosphate. The resulting homologous recombinants were purified by plaque, and clones of HSV-1 17+ 34.5- IL-15ED / S-FLIC-BP-M3 47- were obtained. [Examples]

[0145] Screening of candidate TLR ligands The inventors first began by screening several TLR ligands (i.e., TLR2, 4, or 5) to determine which would be best suited for use in oHSV to treat cancer. Each TLR ligand was inserted into the mammalian expression vector pcDNA3.1. It has been previously shown that the N-glycosylation site inhibits ligand-receptor interaction. Therefore, each gene was mutated to remove the N-glycosylation site, thereby changing the asparagine amino acid to glutamine. In addition, a sequence containing a secretory signal derived from CD33 was added to enable the release of the TLR ligand protein from mammalian cells.

[0146] To screen recombinant TLR ligands, the inventors exposed them to NF-KB reporter systems expressing their target TLR receptors. These systems test the ability of TLR ligands to activate the NF-KB promoter via marker genes. TLR2 ligands were tested on a human TLR2 / NF-κB-AP-1 / SEAP reporter in HEK293 cells (Invivogen) (Figure 2b). TLR4 and TLR5 ligands were screened in 293 cells transiently transfected with the pNL3.2 Luc NF-KB RE / pGL4.54 Luc2 / TK plasmid (Promega) (Figure 2a). From this screening of TLR ligands, the inventors identified FliC_BP as the best candidate (Figure 2a). This result was consistent with FliC_BP's ability to interact with TLR5 on HEK cells and induce activation of the transcription factor NF-κB. This TLR5 ligand offers an opportunity to enhance the antitumor immune response seen in oncolytic viruses through an innate bacterial response. [Examples]

[0147] Design and generation of expression cassettes and oHSVs containing TLR5 ligand and / or IL-15 Next, the inventors constructed a skeletal oncolytic virus based on human HSV-1 (strain 17+) containing the previously described tumor selectivity and immunoregulatory deletions. Referring to Figure 1, a schematic layout of HSV-1 wild-type (WT) is shown (in the upper panel) indicating the corresponding positions of the functionally deleted genes ICP34.5 and ICP47, as discussed below. Next, the inventors inserted the candidate TLR ligand FliC_BP into this skeletal oncolytic HSV virus (oHSV or oHSV GFP) to create a series of vectors expressing either FliC_BP (oHSV-FliC_BP), IL-15 (oHSV_IL-15), or both genes (oHSV-FliC_BP-IL-15).

[0148] ICP34.5 The HSV-1 neurotoxic protein, Infected Cell Protein 34.5 (ICP34.5), is necessary for HSV-1 infection of neurons and other healthy cells because it binds to and blocks the PKR pathway, enabling viral replication. ICP34.5 blocks the activation of the PKR pathway by binding to and activating PP1α phosphatase, which dephosphorylates eIF-2α, thereby preventing the arrest of protein translation by eIF-2. Therefore, for infection of cancer cells, as in the treatment of bladder cancer as described herein, the inventors considered it beneficial for the ICP34.5 gene to be absent, disrupted, or non-functional.

[0149] ICP47 ICP47 is involved in the lytic activity of HSV-1, and therefore, rendering it non-functional enhances that activity. In many cancer cells, the type I IFN signaling pathway is disrupted, and in the absence of ICP34.5, viral replication occurs in cancer cells lacking normal IFN signaling, while normal cells, whose IFN signaling is not impaired, are less tolerant. Thus, we believe that this characteristic of the HSV-1 ICP47 / - mutant provides an important safety mechanism, as infection of normal cells is interrupted in the absence of defects in PKR and type I IFN signaling. Therefore, regarding the infection of cancer cells, for example, when treating bladder cancer, we believe that the absence, disruption, or non-functionality of the ICP47 gene is also advantageous.

[0150] Referring here to the lower panel of Figure 1, a schematic layout of the expression cassette integrated into the disrupted ICP34.5 gene site is shown. The expression cassette contains a nucleotide sequence encoding a flagellum (i.e., FliC BP) derived from Burkholderia pseudomallei, a TLR5 ligand, and its expression is regulated by the CMV promoter. Expression extends in the 5' to 3' direction (left to right as shown in the figure). We found FliC_BP to be a particularly effective candidate for enhancing the antitumor immune response observed in oncolytic viruses through an innate bacterial response.

[0151] In addition, the expression cassette contains a nucleotide sequence encoding the cytokine (IL-15), and its expression is controlled by the RSV promoter. Expression extends in the 3' to 5' direction (right to left as shown in the figure). IL-15 is a pleiotropic cytokine that plays a major role in the development of inflammatory and protective immune responses to invading microorganisms and parasites by modulating immune cells in both the innate and adaptive immune systems. IL-15 expression promotes NK cell proliferation and the cytotoxic function of CD8 T cells. FliC_BP is a bacterial protein that stimulates a potent innate response, and IL-15 and FliC_BP act synergistically together.

[0152] The expression cassette has two adjacent poly(A) tails. The cassette encoding IL-15 and FliC_BP was inserted into oHSV in which the genes ICP34.5 and ICP47 were functionally deleted to construct the vector HSV-1 17+ 34.5- IL-15ED / S-FLIC-BP-M3 47-.

[0153] As described in the following examples, oHSV-1 17+ 34.5- IL-15ED / S-FLIC-BP-M3 47- was then tested for its efficacy in treating cancers such as bladder cancer. [Examples]

[0154] Characterization of oHSV expressing FliC_BP and / or IL-15 in vitro To verify the viral expression of FliC_BP, bladder cancer cell lineage 5637 was infected with oHSV-FliC_BP, and immunostained 24 hours later with mouse polyclonal serum produced against flagellin bacterial protein derived from BP. This staining showed strong FliC_BP expression in the cytoplasm and membrane of infected cells (Figure 2f). No staining signal was detected in oHSV-infected 5637 bladder cancer cells (data not shown) or with secondary antibody alone (Figure 2f).

[0155] To test whether virally expressed FliC_BP can activate the MyD88-NF-KB pathway, the inventors used a TLR receptor NF-KB reporter assay. Supernatant from infected bladder cancer cells was added to 293 cells expressing the TLR5 receptor and transiently transfected with a plasmid containing an NF-KB promoter upstream of the luciferase gene. The oHSV-FliC_BP virus showed a 10- to 18-fold increase in luciferase expression compared to oHSV (Figure 2c). This indicated that secreted FliC_BP produced just 4 hours after infection of bladder cancer cells with oHSV-FliC_BP was able to activate NF-KB in the reporter system (Figure 2c).

[0156] Using a TLR5 inhibitor (αHu TLR5-Fc, InvivoGen), which is a soluble ectodomain of human TLR5, we demonstrated that activation of the MyD88-NF-KB pathway can be partially blocked, and that the activity of virus-produced FliC_BP is at least partially mediated by TLR5 binding. This suggests that the virus-expressed protein can interact with its bacterial counterpart in a similar way and thus is expected to stimulate an innate immune response (Figure 2e).

[0157] To understand the effect of virally expressed FliC_BP alone on macrophage models, THP-1 cells (+PMA) were treated with UV-inactivated oHSV or oHSV FLIC BP. The virus-treated THP-1 cells were incubated for 24 hours, and qPCR was performed for innate cytokine transcripts. The data showed that an enhanced innate immune response was observed with oHSV FLIC BP compared to oHSV (Figure 2g).

[0158] To enhance NK cell activation by the novel FLiC_BP virus, the inventors aimed to include the cytokine IL-15. First, they constructed a virus expressing IL-15 (oHSV IL-15). IL-15 expression from oHSV IL-15 was detected using an IL-15 cell bioassay (Promega). Bladder cancer cell line 5637 was infected with the oHSV-IL-15 virus at an MOI of 0.1 for 48 hours. The supernatant from these infected cells was added to IL-15 bioassay cells engineered to express luciferase in response to IL-15 signaling. This assay demonstrated that biologically active IL-15 was produced from the virus at approximately 30 ng / ml (Figure 2d). IL-15 was added to a virus containing FliC_BP to create oHSV-FliC_BP-IL-15, and its expression was demonstrated in an IL-15 cell bioassay. [Examples]

[0159] In vivo efficacy of oHSV-FliC_BP-IL-15 against bladder cancer The data demonstrate that the oncolytic virus according to the present invention is effective in curing up to 50% of mice in an in vivo subcutaneous model of bladder cancer (Figure 3a, b). After 60 days of remission, these mice were re-challenged with the same bladder cancer cell line MB49 and remained cancer-free (Figure 2c). Splenocytes from cured mice, including CD4, CD8, and NK cells, showed activation of tumor-killing cytokines such as TNF-α in the presence of MB49 tumor cells when infected with oHSV-FliC_BP-IL-15 (Figure 3e, f, g). Splenocytes from untreated mice lacked this activation. [Examples]

[0160] Live, replicated HSV enhances the lysis of bladder tumor cells. methodology 4.2 × 10 6 293 cells in 25 cm² 3The cells were plated into flasks and incubated overnight at 37°C. Next, HSV or HSV5-15 was used to infect cells for 1 hour at an MOI of 1 in 2 ml of 2% FCS medium per flask. HSV5 or HSV5-15 refers to our own nomenclature and to the insertion of FliC_BP into either HSV-1 or HSV-2. After incubation, the virus was removed and 5 ml of 10% FCS medium per flask was added. The cells were then incubated for a further 24 hours. The supernatant was collected, centrifuged at 1500 rpm for 5 minutes at room temperature, and transferred to a 10 cm diameter dish. This cell-free supernatant was exposed to 300 J of UV light (in three fractional doses) and then stored at -80°C.

[0161] Four different transitional cell carcinoma (TCC) bladder cancer cell lines were used according to the table below: KU19-19, VMCUB, and TCCSUP-G cells were used at a rate of 1 × 10⁶ cells per well (100 μL) in a 96-well plate. 4 Individual cells were plated, with 8 × 10⁶ T24 cells per 96-well plate. 3 Individual cells were plated. All cells were incubated overnight at 37°C.

[0162] [Table 1]

[0163] Next, the cells were treated with UV-inactivated and uninactivated HSV / HSV5-15 in fresh 2% FCS medium (50 μL at MOI 0.5) and incubated for 2 hours. 100 μl of fresh 10% FCS medium was added and incubated for 72 hours. The medium was removed, and 100 μl of MTS reagent (CellTiter 96® AQueous Assay, Promega) was added and incubated at 37°C for 1 hour. Absorbance at 490 nm was recorded using an ELISA plate reader.

[0164] result HSV was successfully inactivated by UV irradiation, but a small amount of residual activity remained (see Figure 8).

[0165] Four TCC bladder cancer cell lines (T24, TCCSUP, Ku19-19, and VMCUB) were considered susceptible to oncolysis by HSV5-15 and skeletal HSV. UV inactivation of HSV5-15 and skeletal HSV potently inhibited oncolysis in both viruses and all TCC bladder cell lines tested (see Figure 9).

[0166] These results demonstrate that HSV and HSV5-15, when in a living replication state, not only achieve maximum oncolysis of bladder tumor cells and facilitate direct destruction of cancer cells, but also promote the release of therapeutic proteins such as IL-15 and FliC_BP along with tumor antigens. This release stimulates both innate and adaptive immune responses to tumor antigens and viral proteins, which are essential for resulting in sustained anti-tumor immunity.

[0167] The death of RU19-19 cells at the highest concentrations of UV-inactivated HSV or HSV5-15 is attributed to the residual viral activity after incomplete UV inactivation (Figure 8), combined with the susceptibility of RU19-19 cells to HSV-induced lysis. [Examples]

[0168] FliC_BP expression from plasmids or oncolytic HSV triggers TLR2 activation. The inventors aimed to investigate the effect of FliC_BP on the activation of Toll-like receptor 2 (TLR2) when expressed from both plasmids and oncolytic HSVs.

[0169] methodology NF-KB response SEAP assay for TLR2

[0170] [Table 2]

[0171] Expression of FliC_BP protein from mammalian plasmids 1.5 × 10⁶ per well of a 96-well plate 4 293 cells were plated at a cell density of 1, and incubated overnight until 50–70% confluence was reached. ViaFect® transfection reagent, DNA (7:1 ratio), and diluent (serum-free medium) were warmed to room temperature and gently vortexed. Plasmid DNA (including FliC_BP plasmid and pcDNA EGFP) was added to the diluent Opti-MEM® in proportion. The mixture was gently pipetted to ensure thorough mixing. The ViaFect® transfection reagent was then pipetted directly into the medium containing the diluted DNA, avoiding contact with the plastic tube walls. The mixture was incubated at room temperature for 15–30 minutes. Next, 5 μl of the transfection complex was added to the cells, and the plate was gently shaken on a rotating platform shaker (600 rpm) at low speed for 30 seconds. This process was repeated once more for each sample. The cells were then incubated for 48 hours. After incubation, the supernatant was removed from the cells and centrifuged at 2000g for 3 minutes.

[0172] Expression of FliC_BP protein from oncolytic HSV (HSV5) 1.5 × 10⁶ per well of a 96-well plate 4293 cells were plated at a cell density of 1, and incubated overnight until 50–70% confluence was reached. The cells were then infected with HSV5 or HSV GFP at an MOI of 1 in 2% FCS medium per flask for 1 hour. HSV5 refers to our own nomenclature and refers to the insertion of FliC_BP into either HSV-1 or HSV-2. After incubation, the virus was removed and 10% FCS medium was added to the wells. The cells were then incubated for a further 24 hours. After incubation, the supernatant was removed from the cells and centrifuged at 2000 g for 3 minutes.

[0173] TLR2 stimulation determined using HEK-Blue® detection. HEK-Blue® detection is a specialized cell culture medium designed to detect secreted embryonic alkaline phosphatase (SEAP), which functions as a reporter protein produced and secreted by cells. In the experimental setup, 20 μl of each sample was distributed into the wells of a flat-bottomed 96-well plate, with 6 wells allocated per sample. In addition, 20 μl of 10 ng / ml Pam3CSK4 was added to the control wells designated as the positive control, while 20 μl of sterile growth medium was used as the negative control. To prepare HEK-Blue® hTLR2 cells for the assay, the existing medium was removed, and the cells were gently rinsed in 5–10 ml of pre-warmed PBS in a T-75 flask. Subsequently, 2–5 ml of pre-warmed PBS was added to the flask, and the cells were incubated at 37°C for 1–2 minutes to facilitate detachment. The cells were then detached by gently tapping the flask or using a cell scraper, and all cell clumps were ensured to dissociate by gently pipetting up and down. Importantly, to maintain cell integrity, trypsin was not used for detaching HEK-Blue® hTLR2 cells. After detachment, cells were placed in HEK-Blue® detection medium at a rate of 2.8 × 10⁴ per ml. 5The cells were resuspended at a concentration of approximately 50,000 cells. Approximately 180 μl of cells, equivalent to approximately 50,000 cells, were immediately added to each sample well. Care was taken to avoid prolonged cell incubation at room temperature in HEK-Blue® detection medium, as this can lead to increased background or false-positive readings. The assembled plates were then incubated overnight at 37°C in a 5% CO2 environment to allow for SEAP production and secretion. After incubation, SEAP levels were determined at 600 nm using a plate reader to obtain quantitative measurements of SEAP activity indicating the immune response induced by the tested sample. This assay setup allows for the evaluation of immune activation mediated via the TLR2 signaling pathway and provides valuable insights into cellular responses to various stimuli or treatments.

[0174] result FliC_BP, expressed from either a plasmid or oncolytic HSV, successfully activated TLR2 in the HEK-Blue® hTLR2 assay. This result indicates that FliC_BP can induce a TLR2-mediated signaling pathway, suggesting either direct binding of FliC_BP to the TLR2 receptor or the occurrence of crosstalk between TLR5 (the primary receptor for flagellin) and TLR2.

[0175] Toll-like receptors (TLRs) are crucial components of the innate immune system, responsible for the recognition and response to microbial pathogens. Different TLR ligands can induce unique cytokine profiles via pathway-specific signaling, demonstrating the complexity of TLR pathway interactions. The results show that FliC_BP, a flagellin protein from Burkholderia pseudomallei, can induce the TLR2-mediated signaling pathway, suggesting either direct binding of FliC_BP to the TLR2 receptor or the occurrence of crosstalk between TLR5 (the primary receptor for flagellin) and TLR2. This activity, previously not reported for B. pseudomallei flagellin, or indeed for any bacterial flagellin, may explain the observed enhanced ability of FliC_BP to stimulate the innate immune response. Simultaneous activation of the TLR2 and TLR5 pathways is likely to amplify the production of inflammatory cytokines, thereby contributing to a more robust and immediate immune response. The ability of FliC_BP to activate multiple TLR pathways could be a key factor in its potential use as a therapeutic agent, particularly in the context of oncolytic virus therapy where potent immune activation is desired.

[0176] conclusion The inventors have created a novel oncolytic virus that is effective in BCG non-responder models (and ultimately in patients). Intravesical delivery of this novel oncolytic virus is promising for highly selective tumor infection and cytotoxicity of cancer cells. Advantageously, the virus retains potent tumor cytotoxicity with enhanced immunogenicity by incorporating novel bacterial proteins, along with IL-15, which promotes NK cell proliferation and CD8 T cell cytotoxicity (Figures 2 and 3). Thus, the oncolytic virus can selectively kill cancer cells in the bladder while simultaneously inducing potent anti-cancer immunity to prevent cancer recurrence.

Claims

1. A gene construct comprising a first coding sequence encoding a TLR agonist and a second coding sequence encoding a cytokine.

2. The gene construct according to claim 1, wherein the TLR agonist is a TLR2 agonist, a TLR4 agonist, or a TLR5 agonist.

3. (i) The TLR2 agonist is selected from the group consisting of LpqH, LprA, LprG, LpqT, PhoS1, MPB83, MPT83, LAM, AraLAM, LM, PIM2 / 6, TDM, HSP70, MymA, PE_PGRS33, EsxL, PPE18, PPE26, PPE32, PPE57, Lrp, GPLs, PILAM, PPE60, OmpA, and OmpC; (ii) The TLR4 agonist is FimH; or (iii) The gene construct according to claim 2, wherein the TLR5 agonist is selected from the group consisting of FliC, CagL, CagY, FllA, and FlaB.

4. The gene construct according to any one of claims 1 to 3, wherein the TLR agonist is Burkholderia pseudomallei flagellin protein (FliC_BP).

5. The gene construct according to claim 4, wherein FliC_BP comprises or consists of the amino acid sequence substantially shown in SEQ ID NO: 1 or 3, or a fragment or variant thereof, and / or FliC_BP is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 2 or 4, or a fragment or variant thereof.

6. The gene construct according to any one of claims 1 to 5, wherein the first coding sequence codes for a CD33 secretion signal.

7. The gene construct according to claim 6, wherein the CD33 secretion signal comprises or consists of the amino acid sequence substantially shown in SEQ ID NO: 5, or a fragment or variant thereof, and / or the CD33 secretion signal is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 6, or a fragment or variant thereof.

8. The gene construct according to any one of claims 1 to 7, wherein the cytokine is a chemokine, and optionally the chemokine is selected from the group consisting of CXCL10, CXCL9, CXCL11, CXCL8, CXCL12, CCL2, CCL3, and CCL5.

9. The gene construct according to any one of claims 1 to 8, wherein the cytokine is selected from the group consisting of IL-2, IL-6, IL-10, IL-12, non-secretorized IL-12, IL-15, IL-17, IL-21, IL-33, and type I and type II interferons.

10. The gene construct according to any one of claims 1 to 9, wherein the cytokine is IL-15.

11. The gene construct according to claim 10, wherein IL-15 comprises or consists of the amino acid sequence substantially shown in SEQ ID NO: 9, or a fragment or variant thereof, and / or IL-15 is encoded by the nucleotide sequence substantially shown in SEQ ID NO: 10, or a fragment or variant thereof.

12. A gene construct according to any one of claims 1 to 11, further comprising a first promoter operably coupled to the first coding sequence encoding the TLR agonist, and / or a second promoter operably coupled to the second coding sequence encoding the cytokine.

13. The gene construct according to claim 12, wherein the above or each promoter is selected from the group consisting of CMV, RSV, T7 polymerase, EF1a, SV40, PGK1, Ac5, UBI, MP-84, and MP-135.

14. The gene construct according to claim 12 or 13, wherein the first promoter operably linked to the first coding sequence encoding the TLR agonist is a cytomegalovirus (CMV) promoter.

15. The gene construct according to claim 14, wherein the first promoter comprises or consists of the nucleic acid sequence substantially represented in SEQ ID NO: 12, or a fragment or variant thereof.

16. The gene construct according to any one of claims 12 to 15, wherein the second promoter operably linked to the second coding sequence encoding the cytokine is an RSV promoter.

17. The gene construct according to claim 16, wherein the second promoter comprises or consists of the nucleic acid sequence substantially represented in SEQ ID NO: 13, or a fragment or variant thereof.

18. The gene construct according to any one of claims 1 to 17, wherein the gene construct comprises first and second nucleotide sequences encoding first and second poly-A tails, preferably the first poly-A tail coding sequence is located 3' to the first coding sequence encoding the TLR agonist, and / or the second poly-A tail coding sequence is located 3' to the second coding sequence encoding the cytokine.

19. The gene construct according to claim 18, wherein the first and / or second poly-A tail comprises a nucleic acid sequence substantially represented in SEQ ID NO: 14 or 15, or a fragment or variant thereof.

20. An expression cassette comprising a gene construct according to any one of claims 1 to 19.

21. An oncolytic virus comprising a gene construct according to any one of claims 1 to 19 or an expression cassette according to claim 20.

22. Burkholderia pseudomallei is an oncolytic virus containing a nucleotide sequence that encodes the flagellin protein (FliC_BP).

23. The oncolytic virus according to claim 22, comprising a first promoter operably linked to the nucleotide sequence encoding FliC_BP.

24. An oncolytic virus according to any one of claims 21 to 23, selected from the group consisting of herpes simplex virus (HSV), coxsackievirus, marabavirus, measles virus (MV), Newcastle disease virus (NDV), poliovirus, reovirus, retrovirus, Seneca Valley virus (SVV), alphavirus, Semryqui forest virus (SFV), Sindbisvirus (SINV), vesicular stomatitis virus (VSV), Sindbisvirus (SBV), adenovirus, poxvirus, parvovirus, flavivirus, Zika virus, paramyxovirus, picornavirus, and rhabdovirus.

25. (i) the oncolytic virus is herpes simplex virus (HSV), optionally, the HSV is HSV-1 or HSV-2; and / or (ii) the oncolytic virus is a live replicating virus, according to any one of claims 21 to 24.

26. The oncolytic virus according to any one of claims 21 to 25, wherein the oncolytic virus comprises a functionally deleted ICP34.5 gene, preferably the oncolytic virus comprises at least two copies of the functional deletion of the ICP34.5 gene.

27. An oncolytic virus according to any one of claims 21 to 26, comprising a functionally deleted ICP47 gene.

28. A gene construct according to any one of claims 1 to 19, an expression cassette according to claim 20, or an oncolytic virus according to any one of claims 21 to 27, for use in therapy.

29. A gene construct according to any one of claims 1 to 19, an expression cassette according to claim 20, or an oncolytic virus according to any one of claims 21 to 27, for use in the treatment, prevention, or improvement of cancer.

30. A gene construct, expression cassette, or oncolytic virus for use according to claim 29, wherein the cancer is bladder cancer.

31. A gene construct, expression cassette, or oncolytic virus for use according to claim 29 or 30, wherein the cancer is nonmuscle-invasive bladder cancer (NMIBC) or bladder cancer of BCG-refractory disease.

32. A pharmaceutical composition comprising a gene construct according to any one of claims 1 to 19, an expression cassette according to claim 20, or an oncolytic virus according to any one of claims 21 to 27, and a pharmaceutically acceptable vehicle.

33. A process for producing the pharmaceutical composition according to claim 32, comprising combining a therapeutically effective amount of a gene construct according to any one of claims 1 to 19, an expression cassette according to claim 20, or an oncolytic virus according to any one of claims 21 to 27 with a pharmaceutically acceptable vehicle.

Images